JP2002128684A - Hair grower composition and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a safe hair grower composition having excellent hair- growing effect and slight side effect, containing a hair follicle-derived cell growth activation factor, to provide a method for producing the composition, and to isolate a new strain suitably usable for the method and having the ability to produce the above factor. SOLUTION: This hair grower composition contains, as the active ingredient, a cultured product containing a hair follicle-derived cell growth activation factor which is obtained by culturing microorganisms belonging to the genus Xylaria or Lasiodiplodia having the ability to produce the above factor under such conditions as to produce the factor. The method for producing the composition comprises the steps of culturing the above microorganisms under the conditions mentioned above and obtaining a cultured product containing the factor. The strain, AHU9749 strain (FERM P-17993) or AHU9745 strain (FERM P-17992), each belonging to the genus Xylaria and having the ability to produce the above factor, is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a hair restorer composition, a method for producing the same, and a novel strain used in the method.

[0002]

2. Description of the Related Art In recent years, alopecia such as thinning hair or hair loss has been frequently encountered not only by middle-aged and elderly men but also by young people and women. Alopecia includes alopecia androgenesis, alopecia due to dandruff, seborrheic alopecia, alopecia areata, etc., and the causes are complicatedly related to various causes such as androgen, stress, genetics, and microorganisms. are doing.

[0003] Therefore, conventional hair restorers and hair restorers include components that penetrate into the hair root and expand blood vessels, components that promote blood circulation,
A component that stimulates the nipple and promotes the formation of hair, a component having a bactericidal action, a component that prevents dandruff and Kayumi, a component that gives a refreshing feeling, and the like are blended.

[0004] Specifically, as a hair-growing component, female hormones, vitamin E, pantothenic acid, pepper tincture, cinnamon tincture, assembly extract, cepharanthin,
Photosensitive elements and the like, ethanol and the like as a component having a refreshing feeling and having a bactericidal action, resorcinol, salicylic acid, zinc pyridione and the like as anti-dandruff components, and anti-histamine and the like as a Kayumi prevention component. Many drugs and quasi-drugs have been created. In addition, minoxidil was found to have an excellent hair-growth effect, has already been used as a hair-growth agent in the United States and many other countries, and has recently been launched in Japan.

[0005] However, the conventional hair restorative composition as described above does not provide a sufficient hair restoring effect and sometimes gives an unfavorable irritation to the scalp. Also,
Minoxidil has been reported to cause side effects. Therefore, creation of a safe hair restorer composition having an excellent hair restorer effect and having fewer side effects is desired.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to a hair restorer composition containing a hair follicle-derived cell growth activating factor, which has an excellent hair restorer effect, has fewer side effects and is safe, a method for producing the hair restorer composition, and An object of the present invention is to provide a novel strain having the ability to produce a hair follicle-derived cell growth activating factor that can be suitably used in the production method.

[0007]

The inventors of the present invention have conducted intensive studies and found that AHU belonging to the genus Black Rhinoceros
A belonging to the 9449 strain or the genus Lacio diplodia
It has been found that a substance produced from the HU9745 strain has an excellent hair-growth effect, and that it can exert an excellent effect even in a small amount as compared with the above-mentioned minoxidil, thereby completing the present invention.

[0008] That is, the present invention provides (1) a method of culturing a microorganism belonging to the genus Black Mushroom or the genus Lacio diplodia having the ability to produce a hair follicle-derived cell growth activating factor under conditions that produce the factor. A hair restorer composition comprising a culture containing the factor as an active ingredient, (2)
Cultivating a microorganism belonging to the genus Black Mushroom or L. diplodia having the ability to produce a hair follicle-derived cell growth activating factor under conditions for producing the factor, and a step of obtaining a culture containing the factor. , A method for producing a hair restorer composition comprising a culture comprising the factor as an active ingredient, (3) AHU9749 strain (FERM P- 17993), and (4) AHU9745 strain (FER) belonging to the genus Laciodiprodia having the ability to produce hair follicle-derived cell growth activating factor.
MP-17992).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The hair restorer composition of the present invention has a function of producing a hair follicle-derived cell growth activator (hereinafter, referred to as an activator), and mainly has the ability to produce the activator extracellularly. A microorganism belonging to the genus Xylaria or Lasiodiplodia, preferably an AHU9749 strain (FERM P-17993, hereinafter AHU9749) belonging to the genus Black Rhinoceros
A, which belongs to the genus Lasiodiprodia
HU9745 strain (FERM P-17992; hereinafter,
AHU9745 strain) is obtained by culturing the active factor under the conditions that produce the active factor, and a culture containing the active factor is used as an active ingredient. The active factor has an effect of promoting the growth of hair follicle-derived cells mainly consisting of hair mother cells isolated from hair follicle tissue of mammalian skin, and a culture containing such an active factor as an active ingredient By doing so, the hair restorer composition of the present invention can exhibit an excellent hair restorer effect, and is safe with few side effects. In addition, AHU97 belonging to the black mushroom genus
49 strains and AHU belonging to the genus Lasiodiprodia
9745 is a new strain isolated for the first time by the present inventors. Such strains will be described later.

[0010] In the present invention, the term "culture" refers to a culture obtained by culturing the microorganism under conditions for producing an active factor, or a product subjected to various treatments. It is not particularly limited as long as it includes. As the culture, for example, a culture solution obtained by culturing the microorganism in a liquid medium and removing the microorganism, a concentrated solution obtained by concentrating the culture solution by a known method, or a culture solution obtained by a known method Dry powder obtained by drying can be mentioned. In another embodiment of the culture, the culture solution may be subjected to, for example, an extraction operation using an appropriate extraction solvent such as an organic solvent, or may be a fraction containing the active factor obtained by fractionation by column chromatography. Minutes. Still another embodiment of the culture further includes a culture solution containing the microorganism after the culture of the microorganism or a concentrate thereof, a dry powder, or the like, or a dry powder or cell extract of the microorganism itself after the culture. You may. Since the production of the active factor is also observed in the cells, the desired effects of the present invention can be obtained even when the microbial cells themselves are used as a culture. In addition,
Details of the preparation of the culture will be described later.

From the viewpoint of obtaining the desired effects of the present invention, the content of the culture as an active ingredient in the hair restorer composition of the present invention is preferably calculated in terms of the dry weight of the component contained in the culture. 0.01 to 100% by weight, more preferably 0.01 to 20% by weight, still more preferably 0.1 to 1% by weight.
0% by weight. The content of such a culture can be appropriately adjusted so that a desired effect can be obtained using the hair follicle-derived cell growth activity of the obtained hair restorer composition as an index. The evaluation of such activity can be carried out by an active factor evaluation test described in Test Examples described later.

The hair restorer composition of the present invention is not particularly limited as long as the desired effects of the present invention are not inhibited, and may contain other components in addition to the culture.

[0013] Examples of such other components include known hair growth and hair growth components.
And its derivatives, assembly extract, garlic extract,
Blood circulation promoters such as cepharanthin, carpronium chloride, and acetylcholine, local stimulants such as pepper tincture, cantharistin, tincture tincture, nonylate vanylamide, salicylic acid, resorcinol, keratolytic agents such as lactic acid, placenta extract, pentadecanoic acid glyceride,
Metabolic activators such as pantothenyl ethyl ether, biotin, hinokitiol, allantoin, glycyrrhizic acid,
Antiinflammatory agents such as glycyrrhetinic acid, bactericides such as isopropylmethylphenol, triclosan, zinc pyrithione, hinokitiol, cooling agents such as menthol and camphor, and other female hormones.

Furthermore, alcohols such as ethanol and 1,3-butanediol, polyhydric alcohols, water-soluble polymers, antioxidants, pH adjusters, ultraviolet inhibitors, sequestering agents, thickeners, surfactants Agents, purified water, fragrances, preservatives, antibacterial agents, oils, higher fatty acids, fatty acid esters, humectants, cooling agents, pigments, and other normal cosmetic ingredients, or hormones, vitamins, amino acids, astringents and placenta extract , Elastin, collagen, mucopolysaccharide, aloe extract, loofah water, royal jelly, birch, carrot extract, chamomile extract, licorice extract, salvia extract, altea extract, animal and plant extract components including herbal extract such as yarrow extract The special compounding components described above can be arbitrarily compounded according to the purpose.

The content of these other components in the hair restorer composition of the present invention is not particularly limited as long as the desired effects of the present invention are not inhibited, and the other components used exhibit the desired effects. What is necessary is just to adjust suitably so that it may obtain. If there is a residue, water may be further added to adjust the total amount to 100% by weight. Such water is not particularly limited as long as it does not inhibit the expression of the desired effects of the present invention. For example, ultrapure water, distilled water, ion-exchanged water, purified water, tap water, and the like may be used. it can.

Next, a method for producing the hair restorer composition of the present invention will be described. The method for producing the hair restorer composition of the present invention comprises:
A step of culturing a microorganism capable of producing an active factor under conditions that produce the factor, and a step of obtaining a culture containing the factor. The culture used as an active ingredient in the hair restorer composition of the present invention is preferably obtained by culturing such a microorganism in a liquid medium, since the microorganism used in the present invention mainly produces an active factor outside the cells. It is obtained later by obtaining a culture solution from which the microorganism has been removed.

In the present invention, the microorganism having the ability to produce an active factor may be a microorganism belonging to the genus Black Rhinoceros, preferably AHU9749 strain (FERM P-
17993) and a microorganism belonging to the genus Lacio diplodia, preferably AHU 9745 strain (FERM P-
17992). With respect to the method for producing the hair restorer composition of the present invention, a case where the above-mentioned two novel strains specifically isolated will be described.

(A) Novel strain of the present invention AHU9749 strain and AHU which are the strains of the present invention
All 9745 strains were isolated from plants collected in Malaysia as endophytic fungi. That is, a plant sample was collected, and M.P. Tanaka et al.'S method (Microbes and Environments, Vol. 14, No. 4, 1999)
, A large number of endophytic fungi were isolated from the plant preparation.
Then, the plant endophytic bacteria were incubated at 27 ° C. in a PDY liquid medium.
For 5 days, and the liquid culture from which the cells were removed was subjected to an active factor evaluation test described in Test Examples described later to perform screening. As a result, among the endophytic fungi isolated from the plant at the beginning, substances produced from the two strains exhibited excellent hair-growth effects, and these strains were isolated as the strains of the present invention.

The bacteriological properties of the strain of the present invention are shown below.
When the AHU9749 strain was inoculated on a potato dextrose agar medium and cultured at 27 ° C. for 7 to 10 days, white aerial hyphae developed vigorously, did not form conidia, and had a dark brown thick film in the agar substrate. Form spores. Also,
When inoculated and cultivated in the same manner on a Tzapek-Docs agar medium and a malt extract agar medium, white aerial mycelium grows. However, growth on a Tzapek-Docs agar medium is not very good. When the bacterium is inoculated on a corn-meal agar medium and cultured at 27 ° C. for 7 to 10 days, and further cultured under a fluorescent lamp for 3 weeks, a large number of stromal cells are formed. No sporulation is seen inside the stromal. The strain may be, for example, pH 6,
It grows well under culture conditions at a temperature of 27 ° C.

When the AHU 9745 strain is inoculated on a potato dextrose agar medium and cultured at 27 ° C. for 7 to 10 days, the growth of gray, flocculent mycelia is vigorous, and the medium becomes dark gray to yellow-brown. . Conidia do not form. Also, when inoculated and cultured in the same manner on a Tzapek-Dox agar medium and a malt extract agar medium, the growth of gray, flocculent aerial hyphae is vigorously observed. The strain grows equally well, for example, under the culture conditions exemplified for the AHU9749 strain.

The strain was identified as follows. That is, the strain of the present invention was inoculated into a polydextrose medium and cultured, and M. Tanaka et al.'S method (Microbes and Environments, Vol. 14, No. 4, 1999)
18S rRNA was isolated by
A homology search was performed using the DDBJ / EMBL / GenBank database. As a result, AHU974
Nine strains are Xylaria carphofila
phila) and 99.2% homology, while the AHU9745 strain was 97.9% homologous to Botryosphaeria rodina.
And one of its anamorphs, Lasiodiplodia th
eobromae) with 99.6% homology. The AHU9749 strain is a novel strain belonging to the genus Black Rhinoceros, belonging to the genus Ascomycota, in view of the fact that the morphological characteristics, particularly the formation of sexual spores and conidia, are not observed. Identified.
On the other hand, the AHU9745 strain was identified as a novel strain belonging to the genus Lasiodiprodia from its morphological characteristics.

Although the strain name was not specified for both strains, the strain names were the currently registered 18S rRNA,
This is because the number of sequence data in the ITS region is not yet sufficient, and it is difficult to compare with existing homologous / heterologous strains unless spores of the sexual generation are formed.

The strain of the present invention can produce an active factor even in the cells, but mainly produces the active factor outside the cells. Confirmation that such a strain produces an active factor, and evaluation of the activity of an active factor contained in a culture obtained by culturing the strain, should be performed by subjecting the culture to an active factor evaluation test described below. Can be. That is, a culture is used as a dry powder, a fixed amount of the dry powder is dissolved in ethanol to prepare a sample solution, and (i) in the presence of the sample solution or (ii) the same amount as contained in the sample solution. The cells derived from the hair follicle obtained from the mouse are cultured in the presence of only ethanol, and the number of cells after the culture is counted. Then, a t-test is performed on the number of cells at a risk factor of 5%. As a result, (i)
If there is a significant difference between the number of cells cultured under the condition (i) and the number of cells cultured under the condition (i), the strain produces an active factor; The active factor contained in the culture is determined to have the desired activity.

(B) Culture conditions The medium for any of the strains of the present invention is not particularly limited as long as the strain can grow. Preferably, a medium usually used for microorganisms belonging to fungi or mushrooms is used. For example, PDY liquid medium, F-4
It is preferable to use a culture medium or a liquid medium such as PD broth.

For example, when the strain of the present invention is cultured at pH 6 and a temperature of 27 ° C. for 5 days, the active factor can be sufficiently produced mainly outside the cells together with the growth of the cells. From the viewpoint of promoting the production of the active factor, it is preferable to perform aerobic shaking culture using, for example, a reciprocal shaker (120 rpm, 9 cm amplitude).

(C) Preparation of Culture As described above, the culture used in the present invention is obtained by culturing the microorganism, particularly the strain of the present invention, under conditions for producing an active factor. It is not particularly limited as long as it contains an active factor. For example, a culture solution, a concentrate thereof, a dry powder thereof, or a fraction containing an active factor obtained from the culture solution can be used as a culture. It may also be a culture solution containing the microorganism after culturing the microorganism, a concentrate thereof, a dry powder, or the like, or a dried powder or a cell extract of the microorganism after culturing.

The culture solution is obtained, for example, by culturing the strain of the present invention in a liquid medium as exemplified above, and centrifuging the cells from the culture medium preferably at about 5,000 × g for about 20 minutes. It can be obtained as a supernatant by removing by operation. The concentrate can be prepared, for example, by concentrating the culture solution using ultrafiltration or the like. The dry powder is, for example, freeze-dried culture solution,
It can be obtained by spray drying or the like.

The method for preparing the fraction containing the active factor includes, for example, a method by an extraction operation using an appropriate extraction solvent such as an organic solvent, a method using column chromatography, and the like.

As a method by the extraction operation, for example,
There is a method in which a solvent is added in an amount of 1 to 2 times to a culture solution, and a component fraction extracted by the solvent is obtained by distilling off the solvent. Such a component fraction contains the active factor. Such an operation can be repeated several times to increase the content of the active factor in the component fraction. In addition, the activity evaluation of the active factor in such a component fraction can be performed by an active factor evaluation test described later.

The solvent which can be used in the above-mentioned extraction method is not particularly limited, but from the viewpoint of suppressing the deactivation of the active factor and improving the extraction efficiency, monohydric alcohols, ketones, Organic solvents containing at least one selected from the group consisting of esters, ethers, petroleum ethers, aliphatic hydrocarbons, aliphatic hydrocarbon halides, and aromatic hydrocarbons are preferred. In particular,
C4-8 monohydric alcohols such as n-butanol, n-hexanol, methylamyl alcohol, 2-ethylbutanol and n-octanol; ketones such as isobutyl methyl ketone and methyl-n-propyl ketone; ethyl acetate; acetic acid An ester having 4 to 5 carbon atoms such as isopropyl, an ether having 4 to 8 carbon atoms such as ethyl ether, isopropyl ether, n-butyl ether, petroleum ether, n-
C4 to C8 aliphatic hydrocarbons such as butane, n-pentane, n-hexane, and n-octane; and halides of C1 to C2 aliphatic hydrocarbons such as carbon tetrachloride, chloroform, dichloroethane, and trichloroethylene. ,benzene,
Organic solvents containing one or more of C6 to C7 aromatic hydrocarbons such as toluene are preferred.

As a method utilizing the column chromatography, for example, a combination of silica gel, alumina, synthetic adsorption resin and the like is used. And a method of obtaining the fraction containing the desired active factor by confirming the presence of the activity of the active factor in each fraction by an active factor evaluation test described below.

(D) Preparation of Hair Restorative Composition The hair restorative composition of the present invention comprises the culture obtained according to the above (C) and the other components described above, wherein the content of each component in the hair restorer composition is as described above. It is obtained by blending so as to have the contents and mixing and stirring each component according to a conventional method.

The hair restorer composition obtained as described above can be used as cosmetics, quasi-drugs or pharmaceuticals, and can be used, for example, as a hair tonic, a hair cream or a hair treatment.

[0034]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1: Production of a culture containing an active factor (1) AHU9749 strain (FERM P-1799) was placed on a potato dextrose agar slant medium (Merck).
3), and after incubating for 7 days at 27 ° C., a PDY liquid medium [24 g / L potato dextrose broth (DIFCO 2g / L yeast extract (Oriental Yeast)
, PH 6] and shaking culture at 27 ° C for 5 days (1
20 rpm). The culture medium containing the microorganism after the culture was subjected to a centrifugation operation at 5,000 × g for about 20 minutes to obtain a liquid culture from which the cells were removed.

Example 2 Production of Culture Containing Active Factor (2) AHU9745 strain (FERM P-1799) was added to potato dextrose agar slant slant medium (Merck).
2), and after pre-culture at 27 ° C. for 7 days, agar slices grown with the strain were placed in a 500 mL Sakaguchi flask.
A PDY liquid medium [24 g / L potato dextrose broth (manufactured by DIFCO), 2 g / L yeast extract (manufactured by Oriental Yeast), pH 6] dispensed by 0 mL was inoculated, and shaking culture was performed at 27 ° C. for 5 days. . The culture medium containing the microorganism after the culture was subjected to a centrifugation operation at 5,000 × g for about 20 minutes to obtain a liquid culture from which the cells were removed.

Test Example: Evaluation test for active factor The liquid cultures obtained in Examples 1 and 2 were subjected to an evaluation test for an active factor, and the proliferation activity of mouse hair follicle-derived cells was tested.

(1) Preparation of Sample Solution The liquid culture was freeze-dried to prepare a dry powder, and the resulting powder was obtained so that when added to a test medium described below, the final concentration would be as shown in Table 1. The dried powder was dissolved in 99.5% ethanol. In addition, as a comparative example (positive control),
99. Minoxidil (manufactured by Wako Pure Chemical Industries, Ltd.) so that the final concentration is 70 μg / mL when added to the test medium.
Dissolved in 5% ethanol. The obtained ethanol solutions were used as sample solutions.

(2) Collection of Hair Follicles from Mice The skin of C3H / HeSlc-based neonatal mice 4 days old after birth was aseptically collected and washed several times with 10% FBS-DMEM medium. The muscle tissue was removed, the skin piece was cut into a strip having a width of about 1 mm, and the dermal connective tissue was peeled off so that the lower part of the hair follicle appeared. In order to get as many complete hair balls as possible,
Dermal tissue is further subdivided with a scalpel, and 0.2% collagenase DMEM culture solution (without calcium and magnesium)
After incubating at 37 ° C for 60 minutes, the mixture was cooled to 5 ° C, and the reaction was stopped by adding 10% FBS-DMEM medium.
Hair follicles were collected.

(3) Culture of cells derived from hair follicles The obtained hair follicles were treated with trypsin to obtain hair follicle part cells (mainly hair mother cells), and these cells were cultivated in 10% FBS-DME.
The cells were dispersed in M medium and seeded on a 96-well microplate coated with collagen. After culturing for 24 hours under conditions of 5% CO ₂ and 37 ° C., the culture solution was added to the sample solution or a control medium (negative control) to which 1/100 volume of a 99.5% ethanol-only solution had been added [ MCDB153
In the medium, 5 μg / mL insulin, 5 ng / mL EGF, 0.5 μg / mL hydrocortisone, and 35 μg-protein / mL-bovine pituitary extract (Bovi
nePituitary Extract), followed by culturing for 4 days under the same conditions.
The number of cells was measured using an nting Kit (manufactured by Dojindo). According to such a kit, the number of cells is measured by spectroscopically detecting a specific enzyme activity in a living cell using a coloring reagent, and the absorbance value obtained at that time is proportional to the number of cells.

(4) Evaluation of Cell Proliferation Activity The absorbance obtained in the test medium to which the sample solutions of Examples 1 and 2 and the comparative example were added was compared with the absorbance obtained in the test medium of the control example. The cell growth ratio was calculated for. The significant difference test with the control was performed with a risk ratio of 5% (p
<0.05) was considered significant, and a t-test was performed. Table 1 shows the results
Shown in The cell growth ratio was calculated based on the results (absorbance values) obtained by repeating each experiment of each Example, Comparative Example, and Control Example eight times, and in each case, the average value and the standard deviation of the absorbance values. Then, the average value of each Example and Comparative Example is expressed as a percentage (%) based on the average value of the control example, and each average value (a value expressed as a percentage based on the average value of the control example) ± standard Displayed as deviation.

[0042]

[Table 1]

From the results shown in Table 1, the strain of the present invention, A
Cultures obtained by culturing the HU9749 strain or the AHU9745 strain were significantly (p <0.0
In 5), cell proliferation was observed, indicating that the proliferation activity of hair follicle-derived cells was equal to or better than that of minoxidil at lower concentrations.

Example 3 Production of Hair Restorative Composition Table 2 shows one preferred formulation of the hair restorer composition of the present invention. According to such a formulation example, each component is mixed by a conventional method,
The mixture was stirred to obtain a hair restorer composition.

[0045]

[Table 2]

[0046]

According to the present invention, a novel strain (AHU) having the ability to produce an active factor exhibiting an excellent hair growth effect is provided.
9749 strain and AHU9745 strain). Further, by using a culture obtained by culturing such a strain as an active ingredient, it has an excellent hair growth effect,
A safe hair restorer composition with few side effects is provided.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toshihiro Sakano 5-12, 12 Kengen-cho, Chuo-ku, Osaka Inside Mandom Central Research Institute Co., Ltd. (72) Inventor Mohammed Ismail Abdal Kalim Malaysia 50 Jalan SS7 / 18 Kerana Jaya 47301 Petaling Jaya Serangol F-term (Reference) 4C083 AA031 AA032 AC102 AC122 AC432 AC642 AC852 AD552 AD662 CC37 DD27 EE09 EE22 4C087 AA01 AA02 AA03 BC04 NA05 NA06 ZA92

Claims (6)

[Claims] 1. A method comprising the step of culturing a microorganism belonging to the genus Black Mushroom or the genus Latio diplodia having the ability to produce a hair follicle-derived cell growth activating factor under conditions for producing the factor. A hair restorer composition comprising a culture as an active ingredient. 2. The microorganism belonging to the genus Black Rhinoceros,
The hair restorer composition according to claim 1, which is AHU9749 strain (FERM P-17993). 3. The microorganism belonging to the genus Lasiodiprodia is AHU9745 strain (FERM P-17992).
The hair restorer composition according to claim 1, which is 4. A step of cultivating a microorganism belonging to the genus Black Mushroom or the genus Latio diplodia capable of producing a hair follicle-derived cell growth activating factor under conditions for producing the factor, and a culture containing the factor. A method for producing a hair restorer composition comprising, as an active ingredient, a culture containing the factor, comprising the step of: 5. AHU9749 belonging to the genus Black Mushroom which has an ability to produce a hair follicle-derived cell growth activating factor.
Strain (FERM P-17993). 6. AHU97 belonging to the genus Latio diplodia capable of producing a hair follicle-derived cell growth activating factor.
45 strains (FERM P-17992).